PROJECT SUMMARY Microtubule Severing and Regrowth by Spastin Hereditary spastic paraplegia (HSP) is a neurodegenerative disease that causes progressive gait disorder. The most commonly mutated gene found in HSP patients encodes the microtubule- severing enzyme spastin, which can sever microtubule polymers into shorter fragments. While microtubule severing proteins?spastin, katanin and fidgetin?have been long thought to disassemble the cellular microtubule network, in vivo studies in various organisms have shown that they can actually increase the number of microtubules in cells. Our long-term goal is to establish a framework for how spastin regulates cellular microtubule networks, and how perturbation of spastin activity leads to neuronal degeneration. Recently, by reconstituting the activity of purified spastin, we discovered that the protein possesses a novel activity that promotes the regrowth of severed microtubules. This activity is independent of its canonical severing activity. We showed that the combination of severing and microtubule regrowth promotion can lead to an exponential increase in the number of microtubules and the amount of tubulin polymer. Based on this work, we hypothesize that spastin increases the amount of cellular microtubules by combining these two activities. How spastin perform these functions, however, is poorly understood. The overall objective of this project is to understand the molecular mechanisms of severing and regrowth using a combination of single-molecule fluorescence microscopy, force spectroscopy and mathematical modelling. Our two specific aims are: (i) dissect the molecular mechanics of spastin-dependent microtubule severing, and (ii) test competing models for how spastin promotes microtubule regrowth. Completion of these aims is expected to yield detailed kinetic and mechanical mechanisms for spastin?s activities and to generate reagents that will facilitate future structural and cellular studies, including on the pathophysiology of spastin.